Vpx mediated degradation of SAMHD1 has only a very limited effect on lentiviral transduction rate in ex vivo cultured HSPCs

Abstract

Understanding how to achieve efficient transduction of hematopoietic stem and progenitor cells (HSPCs), while preserving their long-term ability to self-reproduce, is key for applying lentiviral-based gene engineering methods. SAMHD1 is an HIV-1 restriction factor in myeloid and resting CD4+ T cells that interferes with reverse transcription by decreasing the nucleotide pools or by its RNase activity. Here we show that SAMHD1 is expressed at high levels in HSPCs cultured in a medium enriched with cytokines. Thus, we hypothesized that degrading SAMHD1 in HSPCs would result in more efficient lentiviral transduction rates. We used viral like particles (VLPs) containing Vpx, shRNA against SAMHD1, or provided an excess of dNTPs or dNs to study this question. Regardless of the method applied, we saw no increase in the lentiviral transduction rate. The result was different when we used viruses (HR-GFP-Vpx+) which carry Vpx and encode GFP. These viruses allow assessment of the effects of Vpx specifically in the transduced cells. Using HR-GFP-Vpx+ viruses, we observed a modest but significant increase in the transduction efficiency. These data suggest that SAMHD1 has some limited efficacy in blocking reverse transcription but the major barrier for efficient lentiviral transduction occurs before reverse transcription.

Human Dendritic Cell Subsets, Ontogeny, and Impact on HIV Infection

“…Firstly, as outlined above, the earliest studies of DC-HIV interactions could observe infection in vivo . Secondly, the concept that lentiviral restriction factors such as SAM domain and HD domain-containing protein 1 (SAMHD1) do not enable HIV infection solely in DCs is not correct as lentiviral restriction is also observed across many susceptible HIV targets including T cells and macrophages (170, 171). Finally, maturation of DCs can lead to significant blocks in HIV infection and culture/inoculation conditions that favor maturation of DC subsets will likely not reveal any HIV infection.…”

Antiviral immunity and nucleic acid sensing in haematopoietic stem cell gene engineering

“…Importantly, since the restriction is based on the control of dNTP concentration, most of the SAMHD1 restriction is confined to non-proliferating cells, such as macrophages and dendritic cells. In agreement, the relevance of SAMHD1-mediated restriction in HPSC is minimal when these cells are stimulated with growth-promoting cytokines to allow cell survival and proliferation [ 101 ]. However, it is possible that SAMHD1 plays some role in restricting transduction of quiescent, unstimulated HSPC.…”

“…Vpx incorporation does not affect vector titre but renders LV highly efficient in transducing primary human myeloid cells [ 99 ]. This strategy has already been employed to increase the transduction of MDM and DC in relevant preclinical settings [ 124 , 125 ] but only limited benefit has been reported in HSPC thus far [ 101 ]. Coupling Vpx incorporation with early acting transduction enhancers could potentially be beneficial.…”

Viral protein X unlocks the nuclear pore complex through a human Nup153-dependent pathway to promote nuclear translocation of the lentiviral genome

“…Together, these results provided evidence that SAMHD1 degradation activity of Vpx is independent of its interaction with Nup153. Furthermore, Vpx-mediated SAMHD1 degradation alone may not be sufficient for efficient lentiviral infection (Fujita et al, 2012;Li et al, 2015;Baldauf et al, 2017). Nevertheless, it would be interesting to understand whether Vpx-mediated proteasomal degradation of SAMHD1 has any indirect effect on cellular trafficking of the viral genome across NPC.…”